I extracted protein using phenol followed by methanol precipitation. It has the following steps:
* On day 1, I extracted protein using extraction buffer and phenol phase precipitated with ammonium actetae in 100% methanol overnight at -20 C
On day 2, pellet was washed with ice-cold acetone/DTT and the sample was stored at -20C
My question is I have several samples to extract. Can I store just pellet at -20 C. Do I need continue the following step and stored in IEF buffer for long time? If not what is the difference between day 2 and day 3/2D separation
On day 3: Samples were cold acetone extracted via precipitation overnight at -20, the pellet was washed with 50% acetone to remove any residual contamination. Finally the pellet was dried and dissolved in IEF buffer: 8M urea, 4% Chaps, 40mM Tris-Hcl pH 8.5, 100mM DTT and 0.2% ampholyte with trace bromophenol blue (I did bradford assay here) and stored my sample for 2-3 weeks.
What I did here was, I prepared IEF buffer with all above chemicals and aliquot in eppendorf and dissolved my sample in it. Can I store sample for 2 -3 weeks or long time. If not could you please let me know the reason? Can I dissolve sample without DTT and ampholyte? or Do I need to add DTT and ampholyte before rehydrating overnight?
After extracting samples I did sample rehydration with 300microl overnight at room temperature followed by IEF and 2D electrophoresis
Could anyone check and give your valuable suggestions here.